Shiga toxins (Stxs) are critical virulence factors leading to hemorrhagic colitis and the hemolytic-uremic syndrome in humans infected with Stx-producing Escherichia coli (STEC). STEC are NIAID Category B Priority pathogens. In many E. coli strains, the stx genes are encoded on lambdoid bacteriophages, and are transmissible from strain to strain by lysogenic conversion. In preliminary studies, we have demonstrated that aspects of both the initial stages of phage infection and the release of Stx from phage-containing cells occur at the bacterial poles. Based on these findings, we hypothesize that absorption of Stx-encoding phage, injection of phage DNA, and Stx release occur at the bacterial pole and involve pole-specific host factors. These findings predict compartmentalization of specific stages in bacteriophage development and emphasize the importance of bacterial pole physiology and function. In this R21 application, we propose exploratory studies that will test our hypotheses and elucidate the molecular mechanisms underlying the localized uptake and release of bacteriophage by the bacterium. In Specific Aim 1, we will test the hypotheses that Stx-encoding phage bind preferentially to bacterial poles and that phage DNA is injected at the bacterial pole, using state-of-the-art imaging techniques. In addition, because little is known about the molecular mechanisms involved in transit of phage DNA across the peptidoglycan layer and periplasm for any bacteriophage, we will conduct a screen designed to identify bacterial factors involved in these processes. The role of each factor identified in this screen in phage DNA uptake and the localization of any factor shown to be involved will be determined. In Specific Aim 2, we will determine whether release of Stx from the bacterial cell occurs at the pole and will investigate the molecular mechanisms underlying the release of Stx and Stx-encoding bacteriophage from the bacterial cell. Our preliminary results suggest a model in which Stx is transported to the periplasm by a polar process and is then released by localized phage-promoted lysis. We will test this model by evaluating whether S/R induced lysis occurs predominantly at the bacterial pole, by determining the localization of Stx in the periplasm prior to release from the host bacterium, and by identifying, characterizing, and determining the localization of host proteins that are required for these processes. These investigations will lead to insights that will have implications not only for the mechanisms involved in the horizontal transmission of these critical virulence factors by Stx-producing enteric pathogens, but also for the absorption and DNA uptake of a wide variety of bacteriophages and for the physiology of the bacterial pole. [unreadable] [unreadable] [unreadable]